Flow Cytometric Challenges in Plasmacytoid Dendritic Cell (pDC) Identification: Limitation of BDCA-4 (CD304)-Based Gating

Plasmacytoid dendritic cells (pDCs) are a unique subset of dendritic cells specialized in rapid and robust type I interferon (IFN) production, playing critical roles in the pathogenesis and pathomechanisms of many human diseases. Accurate identification of pDCs in peripheral blood mononuclear cells (PBMCs) is challenging due to dynamic and non-exclusive specific expression of surface markers such as blood dendritic cell antigen (BDCA)-2 and BDCA-4. Although BDCA-4 is generally more stably expressed than BDCA-2, prolonged stimulation or inflammatory conditions can induce its expression on multiple non-pDC cell types, reducing the accuracy of pDC identification. Here, we thoroughly investigated BDCA-4 expression dynamics on pDCs and other PBMC subsets following prolonged activation with Toll-like receptor (TLR) 7 and TLR9 agonists. Our flow cytometry analysis revealed a significant increase in BDCA-4-positive non-pDC populations after extended stimulation, primarily corresponding to CD14⁺ monocytes. To overcome this limitation, we performed a gating strategy combining BDCA-4 positivity with a cocktail of non-pDC markers, enabling the exclusion of non-pDCs and accurate identification of pDCs. This approach enables the reliable identification of pDCs within heterogeneous cell populations using only two fluorescent channels in healthy conditions and even during strong activation or pathological states characterized by chronic inflammation.